Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
Salinity-mediated carbonic anhydrase induction in the gills of the
euryhaline green crab, Carcinus maenas夞
Raymond P. Henrya,b,*, Stephen Gehnrichc,b, Dirk Weihrauchd,b, David W. Towleb
a
Department of Biological Sciences, Auburn University, 101 Life Science Building, Auburn, AL 36849, USA
b
Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA
c
Department of Biology, Salisbury State University, Salisbury, MD 21801, USA
d
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL 60612, USA
Received 13 November 2002; received in revised form 7 April 2003; accepted 10 April 2003
Abstract
The euryhaline green crab, Carcinus maenas, is a relatively strong osmotic and ionic regulator, being able to maintain
its hemolymph osmolality as much as 300 mOsm higher than that in the medium when the crab is acclimated to low
salinity. It makes the transition from osmoconformity to osmoregulation at a critical salinity of 26 ppt, and new
acclimated concentrations of hemolymph osmotic and ionic constituents are reached within 12 h after transfer to low
salinity. One of the central features of this transition is an 8-fold induction of the enzyme carbonic anhydrase (CA) in
the gills. This induction occurs primarily in the cytoplasmic pool of CA in the posterior, ion-transporting gills, although
the membrane-associated fraction of CA also shows some induction in response to low salinity. Inhibition of branchial
CA activity with acetazolamide (Az) has no effect in crabs acclimated to 32 ppt but causes a depression in hemolymph
osmotic and ionic concentrations in crabs acclimated to 10 ppt. The salinity-sensitive nature of the cytoplasmic CA pool
and the sensitivity of hemolymph osmoticyionic regulation to Az confirm the enzyme’s role in ion transport and
regulation in this species. CA induction is a result of gene activation, as evidenced by an increase in CA mRNA at 24
h after transfer to low salinity and an increase in protein-specific CA activity immediately following at 48 h posttransfer. CA gene expression appears to be under inhibitory control by an as-yet unidentified repressor substance found
in the major endocrine complex of the crab, the eyestalk.
䊚 2003 Elsevier Science Inc. All rights reserved.
Keywords: Carbonic anhydrase; Crustaceans; Osmoregulation; Salinity
夞 This paper is based on a presentation given in the symposium ‘The comparative physiology of carbonic anhydrase: A tribute to
Dr T.H. Maren’ which took place as part of the American Physiological Society meeting ‘The Power of Comparative Physiology:
Evolution, Integration and Applied’, San Diego, California, USA, August 24–28, 2002. Financial support for the symposium from the
Thomas H. Maren Foundation is gratefully acknowledged.
*Corresponding author. Tel.: q1-334-844-9264; fax: q1-334-844-1465.
E-mail address: [email protected] (R.P. Henry).
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S1095-6433(03)00113-2
244
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
1. Introduction
Marine invertebrates, when acclimated to open
ocean salinity, are osmotic and ionic conformers.
Total osmotic concentration, and the concentrations
of the constituent ions that make up the bulk of
that total, are slightly higher in the hemolymph of
the animal than they are in the surrounding medium. These values, however, are believed to be in
passive equilibrium with those in the ambient
seawater; and if osmotic and ionic concentrations
in the seawater decrease, the concentrations in the
hemolymph decrease in a parallel fashion (e.g.
Florkin and Schoffeniels, 1969; Kinne, 1971).
When osmoconformers are exposed to low environmental salinity they undergo hemodilution and
cell swelling due to the influx of water. They
respond to this challenge with a cell volume
regulatory response in which the concentration of
intracellular solutes is reduced, removing osmotically obligated water, and restoring the cell to near
its original volume (e.g. Pierce and Amende,
1981). The ability of osmoconformers to survive
in low salinity depends in their ability to reduce
the pool of intracellular osmolytes. Most are stenohaline, surviving down to a salinity of approximately 18 ppt, but even the most euryhaline
conformers have a lower lethal salinity in the
range of 8–10 ppt (Kinne, 1971). The species that
can survive salinity fluctuations from full strength
seawater down to fresh water, however, are the
osmotic and ionic regulators, those species that
have the ability to maintain hemolymph osmotic
and ionic concentrations above those in the ambient medium (e.g. Mantel and Farmer, 1983). The
most commonly studied osmoregulators have been
the decapod crustaceans.
With few exceptions, marine invertebrates,
including crustaceans, are conformers in full
strength seawater, and these species undergo at
least some degree of hemodilution when exposed
to low salinity. However, at a critical low salinity,
typically approximately 25 ppt, osmoregulators
activate a suite of cellular and molecular mechanisms that result in the activation of systemic ion
uptake across the primary ion-transporting epithelium, the gills (Towle, 1997; Henry, 2001a,b).
While all ions are believed to be actively transported across the gills in low salinity in these
species, Naq and Cly, the two major ions that
make up over 90% of the total hemolymph osmolality, have received the most study (e.g. Smith
and Linton, 1971; Cameron, 1978; Riestenpatt et
al., 1996). The molecular basis of osmoregulation
involves the coordinated function of a number of
individual transport proteins that are expressed in
the gills. Naq is believed to be transported across
the apical surface of the gill epithelium by one or
more of the following proteins: a sodium–hydrogen exchange protein (NHE) (Towle et al., 1997),
an outwardly directly V-type hydrogen ATPase
coupled to an inwardly directly Naq channel (Lin
and Randall, 1991; Onken and Putzenlechner,
1995), or a Naq yKq y2Cly exchange protein
(Riestenpatt et al., 1996). Cly is believed to move
across the apical membrane via Cly yHCOy
3 or
Cly yOHy exchange (e.g. Kirschner, 1979). The
enzyme carbonic anhydrase (CA), localized in the
branchial cytoplasm, is believed to play a support
role in the general transport mechanisms of all
ions by supplying Hq and HCOy
3 through the
catalyzed hydration of respiratory CO2 as it diffuses through the gills (Henry and Cameron, 1983;
Henry, 1988a,b). Naq is then transported from the
intracellular compartment of the gill into the hemolymph via a basolaterally localized Naq yKqATPase (Towle, 1984; Towle and Kays, 1986),
with Cly following passively down an electrochemical gradient.
A number of the individual transport proteins
appear to be both tissue- and species-specific;
however, the Naq yKq-ATPase, which is believed
to provide the driving force for active ion uptake,
and CA, which is believed to support the active
transport of both cations and anions, are present
in the gills of all euryhaline species. Because of
these features, both enzymes are considered to be
central molecular components of the systemic process of ion transport and regulation.
Among euryhaline crustacean species, the two
that have received the most study are the blue
crab, Callinectes sapidus and the green crab, Carcinus maenas. The green crab is a euryhaline
osmotic and ionic regulator that is routinely found
in estuarine waters of salinities as low as 8–10
ppt and has been acclimated to 5 ppt in the
laboratory (Zanders, 1980; Siebers et al., 1982).
A strong regulator, C. maenas can maintain its
hemolymph osmotic concentration more than 250
mOsm above that in the medium (560 vs. 300
mOsm for crabs acclimated to 10 ppt) (e.g. Zanders, 1980). This is accomplished by high rates of
Naq and Cly influx across the gills (500–800
mmol Naq gy1 hy1, and 250–700 mmol
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
245
Cly gy1 hy1) (Siebers et al., 1987a,b; Lucu, 1989)
that keep the animal in positive salt balance with
respect to low environmental salinity. These high
rates of salt transport require high levels of activity
of both the Naq yKq-ATPase and CA, and these
values are in fact found in the posterior, iontransporting gills of the crab (e.g. Henry et al.,
2002). CA activity, in particular, undergoes an 8fold increase in response to a change in salinity
from 32 to 10 ppt, making this species a good
potential model system for the study of salinitymediated CA induction. This report reviews the
current state of knowledge on the role of branchial
CA in the process of low-salinity adaptation in
decapod crustaceans, and it adds new data on the
regulation of CA expression in the euryhaline
green crab, C. maenas.
2. Materials and methods
2.1. Collection and maintenance of animals
Adult, male intermolt individuals of C. maenas
were collected from the intertidal zone at the
Mount Desert Island Biological Laboratory
(MDIBL) during the months of June and July
(average summer salinitys32 ppt). Crabs were
held in running seawater (32 ppt, 10–12 8C) at
the MDIBL for at least 48 h before use; they were
fed squid twice per week and were starved for 24
h prior to use. For low-salinity acclimation, crabs
were transferred directly into a 120-l recirculating
tank (10 ppt, made by diluting natural seawater
with deionized water produced by reverse osmosis)
equipped with a 15-gallon biological filter. Crabs
were not fed during the acclimation process.
2.2. Experimental protocol
For the experiment involving the use of acetazolamide (Az), a stock solution of 100-mM Az
was made up in deionized water (pH adjusted to
9.0 with NaOH) and titrated to pH 8.2 with 1 N
HCl. Two groups of crabs, acclimated to 32 or 10
ppt for 2 weeks, were used. A small hole was
drilled in the dorsal carapace of the crab, directly
above the heart, and sealed with a rubber septum
and cyanoacrylate glue (Henry and Cameron,
1983). The crabs were weighed, and hemolymph
volume was estimated as approximately 30% of
the wet weight (Gleeson and Zubkoff, 1977).
Control hemolymph samples were taken from the
Fig. 1. Distribution of CA (a) and NaqyKq-ATPase (b) activities in individual gills of C. maenas acclimated for 2 weeks
to 32 ppt (open bars) and 10 ppt (shaded bars).
Mean"S.E.M., Ns5–6. Ts12 8C. Redrawn from Henry et al.
(2002).
sinus at the base of the walking legs using a 22ga needle and 1-ml syringe. An appropriate volume
of the Az stock solution was injected to produce
a circulating concentration of 1 mM. Hemolymph
samples were then taken over a period of 96 h
post-injection. Samples were frozen and stored at
y20 8C until analysis.
For the experiments involving branchial CA
assays, nucleotide sequencing and mRNA expression, crabs were anesthetized in crushed ice for 20
min. Individual pairs of anterior (G3) and posterior
(G7) gills were dissected out. For the simultaneous
measurement of CA activity and mRNA expression, the gills from the left side of the crab were
used in the CA assay, and the gills from the right
side were used for RNA analysis. Anterior (G1-6)
and posterior (G7-9) gills were pooled for the
isolation of the different subcellular fractions of
gill tissue. The crabs were killed by exsanguination.
246
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
To study the presence of a putative CA repressor,
thought to be localized in the major endocrine
complex of the crab, the eyestalk, crabs were
treated with eyestalk ablation (ESA), or a combination of ESA plus injections of eyestalk homogenates. For treatment with ESA, crabs acclimated
to 32 ppt were anesthetized in crushed ice, and
the eyestalks were removed by making a cut where
they attached to the carapace. Crabs were left on
ice for 10 min to allow clotting to occur, and then
they were returned to the seawater system. ESAtreated crabs were left at 32 ppt for 7 days, at
which point they were anesthetized, and the gills
were removed for CA assay. A second set of crabs
were treated with ESA and then injected daily
with homogenates of eyestalks taken from other
crabs that were also acclimated to 32 ppt. A pair
of eyestalks was homogenized in 500 ml of filtered
(0.45 mm) seawater and centrifuged at 10 000=g
for 10 min at 4 8C (Sorvall RC5-B). An individual
crab was given a daily injection of 400 ml of the
supernatant, the equivalent of one pair of eyestalks
per day. Injections were given over a period of 7
days, and the crabs were then anesthetized and the
gills dissected out for CA assay. Sham-operated
crabs were given daily injections of 400 ml of
filtered seawater.
2.3. Analytical methods
2.3.1. Hemolymph osmotic and ionic concentrations
Hemolymph was thawed on ice, sonicated (25
W, 30 s, Heat Systems Microson) to disrupt the
clot, and centrifuged (10 000=g for 60 s, Fisher
235B microfuge) to separate clot from serum.
Total osmolality was determined on 10 ml of serum
by dew point depression using a Wescor 5100C
vapor pressure osmometer. Chloride ion concentration was determined by Ag titration (Labconco
chloridometer), and Naq and Kq concentrations
were measured by flame photometry (Radiometer
FLM3).
2.3.2. CA activity measurements
CA activity was assayed electrometrically using
the delta pH method of Henry (1991a). For the
experiments involving salinity transfers, branchial
tissue from individual gills was homogenized in 2
ml of buffer (225 mM mannitol, 75 mM sucrose,
10 mM Tris, adjusted to pH 7.4 with 10% phosphoric acid) using an Omni 1000 homogenizer.
Samples were centrifuged at 10 000=g for 20 min
at 4 8C (Sorvall RC5-B), and CA activity was
assayed in the supernatant. Protein concentration
was determined by the Coomassie blue dye binding method (Bio Rad Laboratories, Hercules, CA),
and CA activity was reported as mmol CO2 (mg
proteiny1) miny1.
In a separate procedure, pooled anterior or
posterior gills (;5 gm each) were weighed and
homogenized in five volumes of buffer using a
motor-driven teflon-glass homogenizer (minimum
of 16 complete passes) (Henry, 1991b). The homogenates were then subjected to differential centrifugation: 750=g for 20 min; 7500=g for 20
min, and 100 000=g for 90 min, to produce,
respectively, a cellular debris pellet, a mitochondrial pellet, and a final separation of a microsomal
pellet from the cytoplasmic fraction (Henry et al.,
1988; Henry, 1991b). The pellets were resuspended
in buffer, and CA activity was assayed in each of
the fractions as described above. The volume of
each fraction was measured, and raw CA activity
(mmol CO2 mly1 miny1) was multiplied by the
total fraction volume, resulting in values of CA
activity that could be directly compared among
fractions (e.g. Henry et al., 1986).
Cytoplasmic CA activity was titrated against
increasing volumes of a stock solution of 5-mM
Az. The data were transformed and graphed as a
double reciprocal plot (Easson and Stedman,
1937), according to the following relationship:
Io yIsKi y(1yI)qEo
where Eo (the y intercept of the plot) is the total
concentration of free enzyme, Ki is the inhibition
constant, and I is the fractional inhibition of
enzyme activity at an inhibitor concentration of Io.
2.3.3. CA sequencing and RNA analysis
Total RNA from both anterior (G1-6) and posterior (G7-9) gills from crabs acclimated to 35
and 10 ppt salinity was isolated under RNAse-free
conditions (Chomczynski and Sacchi, 1987) by
phenol–chloroform extraction (RNAgents, Promega, Madison, WI). RNA concentrations and
relative purity were measured by UV absorbance.
Single stranded, complementary DNA was then
produced from Poly-A mRNA in 2 mg of total
RNA by reverse transcription using Superscript II
reverse transcriptase (Invitrogen) and oligo dT as
primer.
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
CA amino acid sequences from a variety of
animal species were aligned using Clustal-W, and
highly conserved domains were then used as the
basis for designing degenerate sense and antisense
primers. The putative cDNA for CA was amplified
using a variety of primer combinations and PCR.
Taq polymerase (Red Taq, Sigma, St. Louis, MO)
was added after an initial heating to 92 8C and
cooling to 60 8C. Thermal cycles of 94 8C (1
min), 45 8C (1 min) and 72 8C (2 min) were
repeated a total of 30 times. Later amplifications
employing non-degenerate primers were run at an
annealing temperature of 55 8C rather than 45 8C.
Amplification products were separated electrophoretically on 0.8% agarose gels in 1=TBE
buffer and visualized with ethidium bromide. Individual bands were excised from the gel with
nuclease-free scalpels, and the DNA was extracted
with a spin column kit (Qiagen). Products were
directly sequenced without subcloning using an
ABI 377 or 3100 automated sequencer at the
Marine DNA Sequencing Center, MDIBL. Using
degenerate and Carcinus-specific oligonucleotide
primers for the polymerase chain reaction, we
obtained two partial cDNA sequences that BLAST
searches of GenBank indicated were related to CA
sequences of other species.
To examine the expression of the CA mRNA in
the posterior gills in response to low-salinity exposure, total mRNA was extracted from G7 of green
crabs acclimated to 32 ppt and from crabs during
the acute phase of acclimation (0–4 days) after
being transferred to 12 ppt. Anterior and posterior
gills (G3 and G7) from the opposite side of the
same crabs were also dissected out, immediately
frozen in liquid nitrogen, and stored at y70 8C
until they could be assayed for CA activity. The
change in expression of the CA mRNA was
monitored using a semi-quantitative method of
PCR (Towle et al., 1997). Species-specific primers
were used for amplification of CA sequences; they
are as follows:
81F: GGA GGA AAG CCT TGA GTG GG
82R: CCC TGA ACG TGA AGC AGG AG.
Biotin-dUTP was substituted for a portion of
the dTTP in the reaction mixture. Amplification
proceeded under conditions in which the product
abundance was directly dependent on the availability of cDNA template. Following electrophoresis, products were blotted onto nylon membranes
and the biotinylated DNA was visualized with
247
streptavidin and alkaline phosphatase (New England Biolabs Phototope System). Exposure to Xray film indirectly revealed the relative abundance
of CA mRNA that initiated the reverse transcription reaction. Arginine kinase (AK), an enzyme
that does not change in either activity or expression
in response to salinity, was used as an internal
control (Kotlyar et al., 2000). Cycle-dependency
for both CA and AK was determined (Fig. 10),
and for the semi-quantitative estimation of mRNA,
PCR programs of 18 and 20 cycles were used for
AK and CA, respectively. Protein-specific CA
activity was measured electrometrically as
described above.
3. Results and discussion
3.1. CA as a critical transport enzyme
While the presence of various specific iontransport proteins appears to be tissue- and speciesspecific, CA (along with the Naq yKq-ATPase)
has been found to be present in significant amounts
in all ion-transporting epithelia (e.g. Henry, 1984;
Towle, 1984). As such, CA has always been
believed to play a role in active ion uptake,
especially across the gills of crustaceans; and more
recently it has been characterized as one of the
central components of the suite of biochemical
adaptations that form the basis of the systemic
mechanisms of branchial ion uptake (Henry,
2001a). The two major criteria for this are (1)
salinity-sensitivity of CA expression and (2) measured disruption of ion transport and regulation
when CA is inhibited by Az.
In the green crab, branchial CA activity is
uniform and low in all gills of animals adapted to
32 ppt, a salinity in which they are osmotic and
ionic conformers (Fig. 1a). CA activity undergoes
a salinity-dependent induction, however; and when
crabs are exposed to 10 ppt there is approximately
an 8-fold increase in activity in the posterior three
pairs of gills. This is very similar to the selective
CA induction that occurs in the posterior, iontransporting gills of the blue crab, C. sapidus
(Henry and Cameron, 1982a; Henry and Watts,
2001), and it is believed to be a common adaptive
feature of all euryhaline marine crustaceans capable of osmotic and ionic regulation (Henry, 1984;
Piller et al., 1995). Similar evidence was also
found in euryhaline fresh water species (e.g. salinity-sensitivity of branchial CA activity in the
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R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
ited. C. maenas is a relatively strong regulator,
maintaining a hemolymph-medium gradient of
approximately 260 mOsm when acclimated to low
salinity (10 ppty300 mOsm) (Zanders, 1980, see
also Fig. 2). Both total osmolality and the concentrations of the major hemolymph ions, Naq, Cly
and Kq, are significantly lowered in crabs acclimated to 10 ppt after being given an injection of
1-mM Az (Fig. 3). Osmolality and ionic concentrations were significantly lowered by 6 h postinjection, and they remained depressed through 96
h. Az injection had no effect on hemolymph
osmolality or on the concentrations of the major
ions in green crabs acclimated to 32 ppt (Fig. 3).
This is not surprising, as at this salinity C. maenas
is an osmotic and ionic conformer, and its hemo-
Fig. 2. Osmotic and ionic concentrations in the hemolymph of
C. maenas acclimated to 32 ppt (T0) and transferred to 10 ppt.
Mean"S.E.M. (Ns5–6). Ts12 8C. Redrawn from Henry et
al. (2002).
crayfish, Pacifastacus leniusculus (Wheatly and
Henry, 1987; Henry and Wheatly, 1988).
The increase in CA activity reported here for
the green crab is of similar magnitude to that
reported for other euryhaline crustaceans, but it is
much larger than previously reported (1.5- to 2fold) for C. maenas, given the same low-salinity
exposure (Bottcher et al., 1990). The most probable reason for this discrepancy is the different CA
assays used in the two studies. The pH indicator
dye assay used by Bottcher et al. (1990) is
relatively insensitive, and the large change in pH
(2.7 units) monitored also most likely resulted in
pH-inhibition of the catalyzed hydration reaction
(Coleman, 1980). As a result, it is likely that both
the absolute levels of CA activity and the magnitude of the CA induction were being underestimated by that assay (Henry et al., 2002).
The ability of green crabs to regulate their
hemolymph osmotic and ionic concentrations is
also disrupted when branchial CA activity is inhib-
Fig. 3. Osmotic and ionic concentrations in the hemolymph of
C. maenas acclimated to 32 ppt (open circles) and 12 ppt
(closed circles) and given an injection of Az that would produce a circulating concentration of 1 mM at T0. Mean"S.E.M.
(Ns6). Ts12 8C. Asterisks denote points that are significantly different at the 0.05 level (ANOVA and Tukey’s posthoc test).
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
lymph ion concentrations are maintained passively
in equilibrium with those in the surrounding seawater. This response is similar to that reported in
previous studies of other euryhaline crab species
(e.g. blue crabs), and it supports the general idea
that CA is physiologically important in all crustaceans that are capable of ion regulation. Treatment
of blue crabs with Az had no effect on hemolymph
osmotic and ionic concentrations in crabs acclimated to high salinity (28 ppt) but resulted in a
dose-dependent depression of these values in crabs
acclimated to low salinity (8 ppt) (Henry and
Cameron, 1983; Henry, 1988b). Branchial ion
transport in the fresh water crayfish, Astacus leptodactylus, has also been shown to be Az-sensitive
(Ehrenfeld, 1974), as it has ion transport in blue
crabs acclimated to fresh water (Cameron, 1979).
Inhibition of branchial CA activity inhibits the
active uptake of Naq and also results in a stimulation of Cly efflux, so that the combined effect
is to put the animal into negative salt balance (i.e.
more salts were being lost to the medium by
diffusion than were being taken up by the gills)
(Ehrenfeld, 1974; Cameron, 1979; reviewed by
Henry, 2001a). Furthermore, inhibition of branchial CA with Az in blue crabs acclimated to high
salinity and acutely transferred to low-salinity
results in 100% mortality by 48 h post-transfer, as
a result of the breakdown of the ionic regulatory
process (Henry and Cameron, 1982b).
These results, and the conclusions that branchial
CA was involved in the general ion uptake mechanism of the gills, were called into question by a
study on ion transport in C. maenas that reported
no effect of 100-mM Az on the influx of either
Naq or Cly in isolated, perfused gills of the green
crab (Bottcher et al., 1991). The discrepancy in
the results and conclusions of these studies probably lies in the way Az was used in the two sets
of experiments. Az is slow to permeate into cells;
a circulating concentration of 1 mM in the hemolymph of blue crabs produces full branchial CA
inhibition only after 4 h (Henry and Cameron,
1983); Bottcher et al. (1991) perfused the gills of
green crabs with 100-mM Az for only 20 min, a
concentration and incubation time that most likely
did not result in full inhibition of branchial CA.
3.2. Properties of salinity-mediated CA induction
CA has one of the highest turnover numbers of
any enzyme known (Maren, 1967), and because
of this, it is rarely, if ever, considered to be the
249
limiting step in the biochemical and physiological
processes in which it is involved. CA is also
typically expressed in excess of what is needed to
meet the needs of the processes that it supports.
For these reasons, the salinity-mediated induction
of CA activity is an interesting system to study
for two reasons: (1) it is the largest documented
change in CA expression in the animal kingdom
and (2) it is one of the few examples of a specific
environmental factor (salinity) directly altering CA
expression. Also, different CA isoforms are found
in different subcellular fractions (Henry, 1988b,
1996), and not every isoform responds to environmental changes in the same way.
Salinity-mediated CA induction in crustacean
gills has been localized specifically to the cytoplasmic pool of CA activity, the subcellular fraction that is believed to be involved in ion transport
(Henry, 1988a,b). The pattern of subcellular distribution in the gills of C. maenas turns out to be
very similar to that in the only other crustacean
species studied, C. sapidus. For green crabs acclimated to 32 ppt, anterior and posterior gills had
approximately the same levels of CA activity in
each of the subcellular fractions. The initial pellet
(cellular debris), produced by low-speed centrifugation, contained the highest levels of CA activity
(Fig. 4). CA activity in the microsomal and cytoplasmic fractions were approximately equal, and
both made up less than 10% of the total activity
in the gill homogenate. The distribution of branchial CA in green crabs acclimated to 10 ppt was
altered primarily in the posterior gills, in which
there was a 5-fold increase in activity compared
to posterior gills from crabs at 32 ppt (Fig. 5).
There was no comparable increase in anterior gills.
Most of the measured increase could be traced to
two fractions: cellular debris and cytoplasm. The
increase in cytoplasmic CA activity was almost
20-fold, and this fraction made up approximately
20% of the total branchial CA activity. In contrast,
the microsomal fraction, although it did increase
3-fold over values found at 32 ppt, still made up
only approximately 5% of the total CA activity of
the gill (Fig. 5). CA activity associated with the
cellular debris pellet has been shown to be mostly
soluble (cytoplasmic) CA that is loosely associated
with the pellet and easily removed (Henry et al.,
1988), so the subcellular fraction of CA activity
that is induced in response to low salinity appears
to be localized primarily to the cytoplasmic pool.
In general, it is believed that the cytoplasmic
pool of CA accounts for the overwhelming major-
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R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
Fig. 4. Schematic representation of the procedure for differential centrifugation and separation of the subcellular fractions of the anterior
(AG) and posterior (PG) gills of C. maenas acclimated to 32-ppt salinity. Values for CA activity represent total activity for each
fraction (mmol CO2 mly1 miny1 multiplied by the fraction volume in ml). Values in parentheses indicate percent recovery for each
step. Values are the means of duplicate assays.
ity of the total branchial CA activity, and it is this
pool of enzyme that is induced in response to low
salinity (Henry, 1988a,b), and the data reported
here for green crabs support this conclusion. The
pattern of localization was originally reported to
be different in green crabs. In homogenates of
posterior gills (G7-9) of C. maenas, CA activity
was reported to be highly concentrated in the
microsomal fraction; this putative membrane-associated CA was reported as constituting between 75
and 94% of the total branchial CA activity in
crabs acclimated to 10 and 30 ppt, respectively
(Bottcher et al., 1990). Since this subcellular pool
of CA had been previously described as being
important in CO2 mobilization at the gills (Henry,
1987), it was suggested that branchial CA in C.
maenas was more important in gas exchange and
acid–base balance than in ion regulation. This was
the opposite situation of what had been previously
found for the subcellular distribution of CA activity in the gills of the blue crab, in which 90–95%
of the total branchial CA activity was localized to
the cytoplasmic pool (Henry, 1988a), and the
distribution for C. maenas reported here.
The differences in the results between the study
of Bottcher et al. (1990) and the data reported
here can most likely be traced to alterations in the
procedure of the differential centrifugation used in
the two studies. For the early work on the blue
crab, and for the work reported here, three centrifugation steps were used. The first step, a lowspeed centrifugation, was used to separate intact
cells, nuclei and large cell fragments (cellular
debris). This fraction has been shown to possess a
high level of CA activity that is only loosely
associated with the pellet and that is most likely
cytoplasmic in origin (e.g. Henry et al., 1988).
This step was omitted in the previous work on C.
maenas, and instead, only one centrifugation step,
a single spin of 100 000=g, was used to separate
the microsomal from the cytoplasmic fractions
(Bottcher et al., 1990). This most likely resulted
in the single pellet being made up of a combination
of cellular debris, mitochondria, and microsomes,
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
251
Fig. 5. Schematic representation of the procedure for differential centrifugation and separation of subcellular fractions of anterior (AG)
and posterior (PG) gills of C. maenas acclimated to 10-ppt salinity. Values for CA activity represent total activity for each fraction
(mmol CO2 mly1 miny1 multiplied by the fraction volume in ml). Values in parentheses indicate percent recovery for each step. Values
are the means of duplicate assays.
with the high levels of CA activity coming from
the cellular debris. Thus, the microsomal contribution to the total branchial CA activity was
overestimated.
Interestingly, even though the quantitative level
of CA activity and its contribution to the total
activity of the gill were originally grossly underestimated in C. maenas, the same degree of
increase in CA activity in the cytoplasmic fraction
was seen in response to low-salinity acclimation
in all the studies involving either C. sapidus or C.
maenas. For C. sapidus, the increase in CA activity
in the posterior gills was approximately 13-fold
for the cytoplasmic fraction and 50% for the
microsomal fraction (Henry, 1988a, 1991a), and
the increase for C. maenas was approximately 18and 3-fold for the cytoplasmic and microsomal
fractions, respectively, reported here (Figs. 4 and
5), and 13- and 3-fold, respectively, reported by
Bottcher et al. (1990).
There are two conclusions that can be drawn
from the above review of new and existing data
Fig. 6. Double reciprocal inhibitor titration plots of CA activity
from the cytoplasmic (open circles) and microsomal (closed
circles) fractions of the posterior (G7-9) gills of C. maenas
acclimated to 12-ppt salinity. Regression lines, determined by
the method of least squares, are as follows: cytoplasm (S3):
ys2.71xq21.3 rs0.97; microsomes (P3): ys2.49xq
11.3 rs0.99. Points represent the means of duplicate assays.
252
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
Fig. 7. Partial nucleotide and predicted amino acid sequences of CA-1 amplified from the gills of C. maenas. The 9-amino acid sequence
shown in bold was identified by hydrophobicity analysis.
on branchial CA in euryhaline crustaceans. First,
the subcellular distribution of the enzyme and the
physiological effects of CA inhibition via Az are
consistent with its having a central role in the
support of the general ion-transport process in the
gills of all euryhaline species examined so far.
Second, and perhaps more interesting, is that CA
activity is labile and highly sensitive to environmental salinity, and the increase in CA activity in
response to low salinity appears to be a central
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
253
Fig. 8. Partial nucleotide and predicted amino acid sequences of CA-2 amplified from the gills of C. maenas.
feature of the transition from osmotic and ionic
conformity to regulation.
3.3. CA induction is regulated at the transcriptional level
The large increase in CA activity in response to
low-salinity exposure could be a result of regulatory processes operating at either the transcriptional or translational level. CA induction could be
the end result of gene activation and de novo
synthesis of new enzyme, or the increase in activity
could result from the activation of a large pool of
pre-existing CA. Salinity-mediated CA induction
occurs on the order of days, depending on species
(Henry and Wheatly, 1988; Henry and Watts,
2001; Henry et al., 2002), and that time course is
more consistent with gene activation and protein
synthesis, but direct evidence has been lacking.
One way to approach this question is to see if
differences in CA activity correspond to similar
differences in enzyme concentration.
For the green crab, results from titrating both
the cytoplasmic and microsomal fractions of gill
homogenates from animals acclimated to 10 ppt
indicated that the induction in CA activity was a
result of an increase in CA concentration. The
direct comparison of free enzyme concentration in
the two fractions showed that cytoplasmic CA was
present in roughly twice the concentration as
microsomal CA (Fig. 6); and when the dilution
factor was taken into account for the differences
in total volumes of the two fractions (f10 ml for
cytoplasm and 2 ml for microsomes), the difference in total concentration in the gill was approximately 9-fold. While this is larger than the
approximate 4.5-fold difference in activity between
the two fractions (Fig. 5), it strongly suggests that
differences in activity are due to differences in
enzyme concentration.
The mechanism of CA induction was confirmed
by more direct studies on the expression of the
CA gene. The sequence designated as CA-1 is
likely nearly complete, consisting of 810 nucleotides with an open reading frame coding for a 257amino acid protein (Fig. 7). Although the 59 end
of the cDNA may be complete, the 39 end is not.
Hydrophobicity analysis of the CA-1 amino acid
sequence revealed a 9-amino acid sequence near
the N-terminus (LSLLLVQGA) that could potentially be a transmembrane domain, possibly serving
as a signal sequence targeting the protein to a
specific subcellular region. However, no trans-
254
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
Fig. 9. Multiple alignment of CA-1 and CA-2 partial amino acid sequences from C. maenas (present study) with CA sequences identified
in Tribolodon hakonensis (Acc. AB055617.1), Anopheles gambiae (Acc. AAAB01008807), Aedes aegypti (Acc. AF395662.1), and
Homo sapiens (BC011949.1). Intensity of shading indicates degree of similarity.
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
255
sion (Fig. 11), and continued to increase toward
acclimated levels through 4 days. There was no
change in CA activity in anterior (G3) gills. The
timing and coordination of changes in CA mRNA
and activity indicate that CA induction occurs
through the synthesis of new enzyme, a process
that is stimulated by low salinity and results in an
increase in gene expression in the posterior, iontransporting gills only.
3.4. CA induction is under inhibitory control
Recent work, in print primarily as preliminary
reports, has indicated that CA induction is under
inhibitory control of a repressor substance found
in the major endocrine complex of the crab, the
eyestalk. When green crabs, acclimated to 32 ppt,
Fig. 10. Estimation of mRNA for AK and CA from the gills
of C. maenas acclimated at least 7 days to 35-ppt and 10-ppt
salinity vs. number of cycles used in the PCR reaction.
membrane domains were identified in the remaining portion of the protein, supporting our
suggestion that CA-1 may represent the isoform
that is present in the highest level of expression,
the cytoplasmic isoform.
A second partial CA sequence was identified
and designated as CA-2 (Fig. 8). Multiple alignment of the two partial CA sequences from Carcinus gills revealed similarities to CA sequences
published for other arthropods and even to a
human CA isoform (Fig. 9).
Specific oligonucleotide primers were designed
for semi-quantitative PCR analysis that would
differentiate between CA-1 and CA-2, and these
were used in an analysis of CA mRNA expression
in response to low-salinity exposure. The results
of this experiment were conclusive. There was a
large increase in the expression of CA mRNA in
G7 by 24 h after transfer to 12 ppt (Fig. 11), and
this level of expression persisted through the 4day time course of the experiment. There was no
corresponding increase in CA mRNA in anterior
gills (G3); in fact, it appeared that CA expression
in these gills was actually reduced in low salinity
(Fig. 10), and the expression of AK also did not
change. The initial increase in protein-specific CA
activity in G7 occurred at 48 h post-transfer,
immediately after the increase in mRNA expres-
Fig. 11. Top panel: semi-quantitative estimation of mRNA for
AK and CA from posterior (G7) gills of C. maenas acclimated
to 32 ppt (day 0) and transferred to 12 ppt (days 1–4). Data
shown are representative of five repetitions. Bottom panel: CA
activity in anterior (G3) and posterior (G7) gills of the same
individuals of C. maenas used for the mRNA expression in the
top panel. Mean"S.E.M. (Ns5–6). Ts12 8C.
256
R.P. Henry et al. / Comparative Biochemistry and Physiology Part A 136 (2003) 243–258
Fig. 12. CA activity (mmol CO2 (mg proteiny1) miny1) in
anterior (G-3) (left-hand bars) and posterior (G-7) (right-hand
bars) gills of C. maenas. (a) Control crabs acclimated to 32ppt salinity. (b) Crabs acclimated to 32 ppt and injected with
filtered seawater once a day for 7 days. (c) Crabs acclimated
to 32 ppt and treated with ESA and left for 7 days. (d) Crabs
acclimated to 32 ppt, treated with ESA and given an injection
of eyestalk extract once a day for 7 days. Mean"S.E.M. (Ns
5–8). Ts12 8C.
were treated with ESA, CA activity in the posterior
gills (G7) increased by 50% over 7 days (from
;200 to 300 mmol CO2 (mg proteiny1) miny1),
while there were no changes in CA activity in
anterior gills (Henry et al., 2000; see also Fig.
12). However, when crabs acclimated to 32 ppt
and treated with ESA were then given daily injections over a 7-day period of the supernate of
homogenates of eyestalks taken from other crabs
at 32 ppt, the ESA-stimulated CA induction was
abolished (Fig. 12). Injections of filtered seawater
had no effect on CA activity in either anterior or
posterior gills.
Furthermore, ESA appeared to potentiate the
normal low-salinity-mediated CA induction. In
crabs acclimated to 32 ppt, treated with ESA, and
then transferred to 12 ppt, CA activity in the
posterior gills increased to a greater degree
(f20%) than in untreated crabs given the same
salinity transfer. In the stenohaline crab, Cancer
irroratus, however, CA induction did not occur in
any gills in response to low-salinity (18 ppt)
exposure, and ESA had no effect on branchial CA
activity in those crabs at either high or low salinity
(Henry et al., 2000). These early results indicate
that there is a substance in the eyestalk that acts
as a repressor of CA expression, keeping the levels
of CA activity in the gills low in animals acclimated to high salinity. When the eyestalks are
removed, a degree of CA induction occurs even in
the absence of a low-salinity stimulus. The effects
of this putative repressor are removed during
acclimation to low salinity, resulting in an increase
in CA gene expression and the observed high
degree of CA induction. This idea is further
supported by the fact that injections of extracts of
homogenates of eyestalks from crabs acclimated
to high salinity reduce CA induction by approximately 50% in intact crabs and by 70% in crabs
treated with ESA (Henry, 2001b). The specific
localization of the repressor within the eyestalk,
its chemical nature and composition, and the nucleotide sequence of the gene that encodes it are
currently under investigation.
Acknowledgments
Results of original work present here were
obtained primarily at the MDIBL with support
from the Salisbury Cove Research Foundation
(RPH and SG). RPH and DWT were supported
by the National Science Foundation (IBN 0230005 and IBN 98-07539, respectively). SG was
also supported by an NSF Research Opportunity
Award.
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